The five classes of white blood cells or leukocytes normally found in whole blood samples are neutrophils, lymphocytes, monocytes, eosinophils, and basophils. To determine the relative proportions of these five normal types of white blood cells, as well as to detect the presence and concentration of any abnormal cells in a whole blood sample, a medical diagnostic procedure is conventionally performed to examine a dried, stained smear of blood on a microscope slide. Such a procedure is referred to as a differential white blood cell count and is described in Miale, J. B., "Laboratory Medicine--Hematology", (1967), C. V. Mosby Company, St. Louis, Mo., pp. 822-830, 1126, 1127 and 1130. In addition to the above-listed five classes of leukocytes in blood samples, differential white blood cell counts also detect and measure large unstained cells ("LUCs"). LUCs represent a small fraction of white blood cells in normal blood samples and comprise such cell types as large lymphocytes, activated lymphocytes, plasma cells, blast cells, and peroxidase-negative monocytes, neutrophils, and eosinophils.
Semi- and fully-automated hematology processes and automated flow system apparatuses therefor have been developed to ease the burden of differential white blood cell counting and blood sample analyses, such as described in U.S. Pat. No. 3,741,875 to Ansley et al.; U.S. Pat. No. 4,099,917 to Kim; and U.S. Pat. Nos. 4,801,549 and 4,978,624 to Cremins et at. Such processes and systems use electro-optical and cytochemical procedures to specifically detect, identify, quantify, and label individual cell types. In addition, manual procedures for determining white blood cell differential counts are known in the art; for example, see Miale, J. B., "Laboratory Medicine--Hematology", (1967), C. V. Mosby Company, St. Louis, Mo.
U.S. Pat. No. 5,389,549 to Hamaguchi et at. describes methods and reagents used for classifying leukocytes, in which the methods involve the detection of changes in electrical impedance at high frequency or the differences in conductivity between particles and fluid medium, and the reagents require one or two component solutions which contain specific types of anionic and nonionic polyoxyethylene-based surfactants having 18-30 repeating oxyethylene units per molecule and which also contain hyper- or hypo-osmotic agents and solubilizing agents. The two component reagents require both a first liquid diluent fluid and a second lysing reagent fluid.
An earlier procedure for preparing a cell suspension for use in such systems comprised treating an uncoagulated blood sample with a surfactant for about 1.5 minutes to precondition the red blood cells for lysis; thereafter adding a fixative to the cells for about 1 minute while maintaining a neutral pH; and then incubating the mixture at about 58.degree. C. to 60.degree. C. for about 2 minutes to lyse the red blood cells and fix the white blood cells, as described in U.S. Pat. No. 4,099,917 to Kim.
U.S. Pat. Nos. 4,801,549 and 4,978,624 to Cremins et al. describe a method and reagent for the determination of a differential white blood cell count which is performed more rapidly, which lyses red blood cells in a whole blood sample without damaging the white blood cells, and which causes minimal extra-cellular precipitation or clumping of cells. Such precipitates or cell clumps generate ambiguities in the cell detection and recognition phase(s) of the procedure. The procedure (or "peroxidase method") described involves a mixture comprising peroxide (i.e., hydrogen peroxide) and a suitable chromogen to stain and differentiate particular cell types in the leukocyte class.
It is imperative in each of these processes that as many red blood cells as possible be lysed, since red blood cells outnumber white blood cells by about 1000-fold in normal blood. Because of this, even if one percent of the red blood cells remains unlysed, it is difficult to achieve an accurate and precise white blood cell differential count.
In the peroxidase method as disclosed in U.S. Pat. Nos. 4,801,549 and 4,978,624, red blood cells are lysed and white cells are crosslinked or "fixed" after a whole blood sample is mixed with a solution comprising only one surfactant, a fixative such as paraformaldehyde or formaldehyde, a sugar or sugar alcohol, and a buffer to maintain approximately neutral pH. Hydrogen peroxide and an electron donor chromogen, such as 4-chloro-1-naphthol, form a dark-purple-colored precipitate in the peroxidase-positive granules located in the cytoplasm of certain white cells, namely, neutrophils, eosinophils, and monocytes. The precipitate is an insoluble reaction product the formation of which is catalyzed by endogenous peroxidase enzymes in the intracellular granules. Differentiating the cell types is carded out by electro-optical analysis in which cell size and degree of staining are measured (i.e., forward angle scatter versus absorbance) on a cell-by-cell basis and plotted in a cytogram which is then analyzed to obtain both a total white cell count and differential count of the different types of white cells in the sample. In addition, the total white blood cell count can be obtained independently of the differential count.
Prior to the improvements and advantages afforded by the present invention, an alkaline peroxidase diluent had been described and particularly used in an alkaline peroxidase method of white blood cell classification carded out on a Technicon H6000.TM. automated analyzer system. The prior alkaline peroxidase diluent had major drawbacks, such as instability of the reagent components and a consequent short shelf and storage life. In addition, the user was required to prepare a homogeneous working solution of the alkaline peroxidase diluent in order to carry out the alkaline peroxidase method. This was accomplished by the user's having to mix together a solution containing a high level (i.e., 4.5%) of sodium dodecyl sulfate and a solution containing a high level (i.e., 30%) of Brij.RTM. 35, thereby resulting in a working alkaline peroxidase diluent having elevated concentrations of the two classes of surfactants. Such user preparation not only involved irritant diluent reagent components, but was also laborious, and had to be performed a number of times, because the resulting homogeneous working alkaline peroxidase diluent was stable for only one week. As will become clear, the instability, the short storage capacity, and the user-handling problems of the alkaline peroxidase diluent, as well as the commercial disadvantages related thereto, have been vastly improved upon and overcome by the present invention as described herein.
Also prior to the present invention, a major drawback which hampered the accuracy and reliability of cell separation and quantification methods, particularly the peroxidase method of leukocyte differential counting, was variable rinse carryover in the method. It is known that rinse carryover varies from system to system and from analysis to analysis. In particular, the accuracy and precision of leukocyte differential analyses based on the peroxidase reaction method frequently suffered from the effects of such variable rinse carryover in the method. Those skilled in the art have assumed that rinse carryover contributes only a volumetric dilution to the method and that rinse carryover plays no active or functional role in the reaction steps of the method. Indeed, until the inventive discovery of the present invention, the skilled practitioner did not realize that rinse carryover was more than a simple volumetric effect in blood sample analysis, and had no solution to the problems offered by the present invention and described herein.
In addition, there is a need in the art for improved reagents and methods for analyzing and extracting useful clinical information from both fresh (i.e., less than or equal to eight hours postdraw) blood samples and also aged blood samples that may have been stored for up to about 48 hours at room temperature. It is also necessary to develop the appropriate reagent solutions and compositions comprising components which will alleviate the newly-described problems generated by variable rinse carryover and will yield accurate and reliable results, especially, but not limited to, in the employment of electro-optical analysis of a variety of blood sample types, e.g., fresh whole blood samples, aged whole blood samples, abnormal whole blood samples (e.g., hospital or patient source), and normal whole blood samples (e.g., non-hospital or "healthy" donor source) stored in a variety of ways (e.g., in the cold or at room temperature). There is a further need in the art for reagent compositions that are very stable, have long shelf and storage lives (e.g., greater than one week), and require no user or customer preparation or handling prior to their use in carrying out leukocyte differential counting methods and obtaining results therefrom. It is also necessary to achieve and/or to maintain acceptable levels of noise at the origins of the resulting cytograms when carrying out differential counts on room temperature-stored or aged blood samples using automated analyzers.